Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient

Abstract

The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism.We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making it broadly available to the public health and research communities. We hope that open access to this important reagent will expedite development of medical countermeasures. BACKGROUNDA novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the source of a pneumonia outbreak in Wuhan China in late 2019 (1, 2).The virus was found to be a member of the beta coronavirus family, in the same species as SARS-CoV and SARS-related bat CoVs (3, 4). Patterns of spread indicate that SARS-CoV-2 can be transmitted person-to-person, and may be more transmissible than SARS-CoV (5-7). The spike protein of coronaviruses mediates virus binding and cell entry. Initial characterization of SARS-CoV-2 spike indicate that it binds the same receptor as SARS-CoV, ACE2, which is expressed in both upper and lower human respiratory tracts (8). The unprecedented rapidity of spread of this outbreak represents a critical need for reference reagents. The public health community requires viral lysates to serve as diagnostic references, and the research community needs virus isolates to test anti-viral compounds, develop new vaccines, and perform basic : bioRxiv preprint and described its genomic sequence and replication characteristics. We have made the virus isolate available to the public health community by depositing into two virus reagent repositories. RESULTS and DISCUSSIONA patient was identified with confirmed COVID-19 in Washington State on January 22, 2020 with cycle threshold (C t s) of 18-20 (nasopharyngeal(NP)) and 21-22 (oropharyngeal (OP))(1). The positive clinical specimens were aliquoted and refrozen inoculation into cell culture on January 22, 2020. We first observed cytopathic effect (CPE) 2 days post inoculation and harvested viral lysate on day 3 post inoculation ( Figure 1B and 1C). Fifty µl of P1 viral lysates were used for nucleic acid extraction to confirm the presence of SARS-CoV-2 using the CDC molecular diagnostic assay (1). The C t s of three different nucleic acid extractions ranged from 16.0-17.1 for N1, 15.9-17.1 for N2 and 16.2-17.3 for N3, confirming isolation of SARS-CoV-2.A C t of less than 40 is considered positive. The extracts were also tested for the presence of 33 additional different respiratory pathogens with the fast track 33 assay. No other pathogens were detected. Identity was additionally supported by thin section electron ...

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